In the arena of mammalian reproduction, many diagnostic and therapeutic procedures exist to aid the reproduction practitioner in making a diagnosis and choosing an appropriate course of action. In mammalian embryology, the mammalian oocyte enters the first meiotic division during fetal life, but becomes arrested in late prophase (in the dictate or diffuse diploid stage of meiosis) before or just after birth (Beaumont, H. M., et al., Proc. R. Soc. London (Series Biological Sciences) 155:557-579 (1962). Resumption of meiosis normally does not occur until shortly before ovulation when previously unidentified growth factor trigger ovarian development, followed by a surge of gonadotropins prompts the resumption of meiotic maturation (Dekel, N., et al., Proc. Nat'l Acad. Sci. U.S.A., 75:4369-4373 (1978).
Currently infertility in humans ranges from approximately 10-15% of couples and the risk of infertility is doubled for women between the ages of 35-44 as compared to women between the ages of 30-34. In the United States, the majority of infertility can be accounted for by problems in the female. is a basement membrane protein that must be synthesized and secreted as the follicle grows. It is another marker specific for granulosa cell differentiation and development in early stages of follicular growth. Another study that was published by Vinter-Jensen et al., 1995 looked at the treatment of mini-pigs with EGF. In these studies it was found to stimulate growth of heart, liver, and urinary tract. However, reproductive organs were not closely evaluated in these studies. Other indirect support has been shown in different species. Eppig and O'Brien published in 1996 that treatment with EGF of neonatal mouse ovaries collected and cultured in vitro, increased the number of eggs recovered for in vitro maturation and fertilization. Again this is consistent with our hypothesis that EGF enhances follicular development. Another study published in 1996 by Breider, et al., the investigator looked at the effect of EGF on mature rats. In this study EGF was intravenously infused for a four-week period into mature rats and multiple organs and tissues were evaluated. Overall, EGF stimulates growth and proliferation of many tissues. Specifically in the ovaries of these animals, the ovarian weights were increased which was accompanied by the increase in the number of corpora lutea (CL) found in these ovaries. The CL is a structure that results from the ovulation of a mature antral follicle. This means that in the ovaries there were increased numbers of ovulations. Again, these studies were done in live animals demonstrating the feasibility of using in vivo treatments with EGF.
Epidermal Growth Factor is a peptide hormone that stimulates the growth and differentiating of epidermal tissues during embryogenesis (Carpenter, G., et al., Exper. Cell. Res. 164-1-10 (1986); Kris R. M. et al., Bio-Technol. 3:135-140 (1985)). EGF may be purified from natural sources or may be obtained through application of recombinant DNA technology.
EGF is a 53-residue polypeptide (M, -6000) that is mitogenic for a variety of cell types both in vivo and in vitro (Carpenter & Cohen, 1979). EGF was originally purified from the male mouse submaxillary gland (SMG) (Cohen 1962) and subsequently from human urine (Cohen & Carpenter, 1975; Gregory, 1975). Antibodies raised against mouse or human EGF are used to confirm expression in tissues or body fluids using immunoassays or immunocytochemical staining. Highest levels of EGF have been found in SMG (mouse), kidney, pancreas, duodenum, urine and milk (see Carpenter, 1985; Gregory, 1985; Burgess, 1989; Fisher & Lakshmanan, 1990). However, there is little information regarding EGF expression in other species because antisera against EGF show very little cross-species reactivity (Gregory, Holmes & Willshire, 1979; Schaudies & Savage. 1986) necessitating the development of homologous immunoassays (Joh. Itoh, Yasue et al. 1989). Despite their limited immunological cross-reactivity, both mouse and human EGF bind to cellular receptors on various cell types from several species with very similar affinities and efficacy (see Carpenter & Cohen, 1979; Carpenter, 1987). While heterologous radioreceptor assays are therefore possible, they lack specificity since other polypeptides (e.g. transforming growth factor alpha (TGF-alpha) are known to bind to the same receptors (see Burgess, 1989; Massague, 1990). In addition, indirect modulation of EGF receptor affinity by heterologous ligands has been widely reported (see Schlessinger. 1986).
A homologous radioimmunoassay for the measurement of EGF levels in pig tissues and body fluids has been developed using an antiserum to recombinant porcine EGF. The assay is highly specific, showing no cross-reactivity with a variety of other polypeptides including the structurally related protein, transforming growth factor-alpha. Furthermore, &lt;1% cross-reactivity was observed with mouse EGF emphasizing the necessity for homologous assays for EGF measurement. Immunoreactive EGF was present in extracts of pig kidney and pancreas (3.44+1-0.43 and 0.76.+-.0.13 (S.E.M.) pmol/g wet weight respectively), but was not detected in extracts of submaxillary gland or liver. Although immunoreactive EGF was not detectable in uterine, allantoic or ovarian follicular fluids, colostrum contained EGF at biologically active concentrations. Immunoreactive EGF was also present in pig urine, with similar concentrations in samples from male or female animals. In addition, pig urine inhibited the binding of I-labeled EGF to 3T3 fibroblasts and stimulated DNA synthesis in quiescent monolayers of these cells, indicating that the immunoreactive material in urine is biologically active. Quantitative comparisons of the data presented here with that published previously indicate considerable species variation in the EGF levels of various tissues and body fluids.
In follicular development, the development of a dormant primordial follicle into a large mature follicle must occur before the ultimate mature follicle is stimulated to ovulate and produce fertilized eggs. This general process is the key for the production of eggs in many species. The ovary is basically a reservoir of dormant follicles and through the process of follicular development, some of these dormant follicles will develop and mature to subsequently produce hundreds to thousands of eggs. Follicular development can be envisioned as a pipeline and the control of the process consisting of many valves or potential regulatory steps. In the later stages of follicular development, it is known that Follicle Stimulating Hormone (FSH) is important for growth and development of mature antral follicles and ultimately Luteinizing Hormone (LH) stimulates the ovulation of a mature follicle and the production of the egg. However, the factors regulating early steps of follicular development have been unknown.
The present invention provides understanding and procedural methods of how some of these early regulatory points are controlled in ovarian follicle development and have applications to mammals and vertebrate animals. For example, methods of the present invention applies to bovine, equine, porcine, canine, feline, human mammals, birds, fish, reptiles and the like. Applications of this technology will benefit farm animals, humans, endangered species, zoo animals and farmed birds and fish.